HPLC columns

High-performance liquid chromatography (HPLC) is a widely used analytical technique for separating and analyzing chemical compounds in various samples. The heart of an HPLC system is the chromatography column, which is filled with a stationary phase that interacts with the analytes to achieve separation. The choice of column chemistry is critical to the success of an HPLC analysis, as it determines the selectivity, efficiency, and resolution of the separation.

Reverse phase columns are a common type of chromatography column used in high-performance liquid chromatography (HPLC) and other liquid chromatography techniques. They are widely employed for the separation and analysis of a wide range of organic and hydrophobic compounds. The term "reverse phase" refers to the nature of the stationary phase, which is nonpolar, while the mobile phase is polar. This reversed polarity compared to normal phase chromatography allows for the separation of molecules based on their hydrophobicity and other related properties.

Here are some key features and aspects of reverse phase columns:

Stationary Phase: The stationary phase in reverse phase columns is typically composed of silica particles or other materials that have been modified to introduce nonpolar or hydrophobic functional groups. The most common modification is the bonding of octadecyl (C18) or octyl (C8) groups to the silica surface, but other variations exist, including C4 (butyl) and C2 (ethyl) phases. The choice of the specific hydrophobic group influences the separation characteristics.

Why Silica? 

Mobile Phase: The mobile phase in reverse phase chromatography is typically a polar solvent or a mixture of solvents. Common mobile phase compositions include water and various organic solvents like methanol or acetonitrile. The choice of mobile phase composition is crucial for achieving the desired separation, as it influences the elution order and retention time of analytes.

Separation Mechanism: In reverse phase chromatography, analytes are retained on the column based on their hydrophobic interactions with the stationary phase. Hydrophobic compounds tend to interact more strongly with the hydrophobic stationary phase, leading to longer retention times. As the polarity of the mobile phase increases (e.g., by increasing the organic solvent content), analytes are eluted from the column in order of increasing hydrophobicity.

Applications: Reverse phase columns are widely used for a broad range of applications, including pharmaceutical analysis, environmental monitoring, food and beverage analysis, and biochemistry. They are particularly effective for the separation of small organic molecules, such as drugs, pesticides, natural products, and metabolites.

End-Capping: Some reverse phase columns are "end-capped" to block residual silanol groups on the silica surface. This end-capping helps improve peak shape and minimize secondary interactions that can lead to peak tailing.

Particle Size: HPLC columns have different pore sizes. The pore size is used according to the molecule size of the substance to be analyzed on the column. The small molecule size substances are analyzed on 8-12 nm pore size columns. Usually, 5-10 µm particles are used in pharmaceutical product testing. A small pore diameter means the larger surface area of packing particles in the column. Larger pore sizes have a small surface area of packing material of column. The surface area of the packing particles is inversely proportional to the pore diameter of the column.

Selectivity: The selectivity of a reverse phase column can be adjusted by changing the mobile phase composition, temperature, and the specific type of stationary phase (e.g., C18, C8, C4). These adjustments can be used to optimize separations for specific analytes and applications.

In summary, reverse phase columns are a versatile and widely used tool in chromatography, offering effective separation of hydrophobic compounds. They play a crucial role in various fields, including pharmaceuticals, chemistry, and life sciences, where the separation and quantification of organic molecules are essential.

HPLC COLUMN CHEMISTRY

There are several different types of column chemistry commonly used in HPLC, including:

Reverse Phase (RP): In reverse phase chromatography, the stationary phase is non-polar and the mobile phase is polar. This type of column chemistry is most commonly used for the separation of polar and hydrophobic analytes, such as drugs, peptides, and lipids. The stationary phase is typically made of silica particles coated with a hydrophobic layer, such as C18 (octadecyl) or C8 (octyl) groups. Reverse phase columns are widely used in pharmaceutical, environmental, and food analysis.

Why Silica? 

Silica is a common material used as the stationary phase in High-Performance Liquid Chromatography (HPLC) columns. The stationary phase in an HPLC column is responsible for separating analytes based on their interactions with the stationary phase and the mobile phase. Silica, specifically in the form of silica gel or silica-based particles, is widely used in HPLC columns due to its many advantages:

High Purity: Silica gel used in HPLC columns is typically of high purity, which reduces the risk of contaminating or altering the sample being analyzed.

How can silica gels be classified for HPLC?

Silica gels can be differentiate according to many criteria. Three of these are mentioned below and briefly explained.

1. According to their particle shape

Nowadays, silica gel is almost perfect spherical. There are only a few materials, which are based on broken silica gel, e. g. μBondapak™ (Waters™) or Lichrosorb™ (Merck™).

Compared to irregularly shaped particles, spherical particles have the advantage that they exhibit a higher efficiency with low back pressure at the same average particle size.

2. By Type-A, -B and -C

The so-called Type-A silica gel was the first silica gel used for HPLC. It contains a large proportion of ionized (acide) silanols, which lead to undesirable peak shapes and in some cases insufficient separation performance.

Nowadays, high-purity silica gel is almost used for chromatography applications, which is also referred to type B silica gel. Compared to type A silica gel, this shows only minimal proportions of ionized silanol groups. This increases the pH stability of the silica gel and the separation efficiency is improved.

Type-C Silica Gel™ was developed by Prof. Joseph Pesek at the San Jose State University, USA. This Silica gel is today marketed by the company MicroSolv™ Technology Corporation. It is based on type-B Silica gel. The otherwise existing free silanols are replaced by silica hydrides. As a result, unwanted analyte-silanol interactions are almost completely suppressed and no hydrate shell is formed with water.

3. According to their modification

Unmodified silica gel carries polar silanols and siloxanes on the surface. For almost all types of chromatography, certain chemical groups are bound to these polar groups in order to modify the surface properties of the silica gel. Nowadays, a variety of HPLC columns with differently modified silica gels are available. Octadecyl-modified silica gel is still the most used stationary phase.

Chemical Inertness: Silica is chemically inert, which means it does not react with most analytes or mobile phase solvents. This inertness is essential for maintaining the integrity of the sample and preventing unwanted chemical reactions.

Adjustable Surface Chemistry: Silica can be modified to introduce specific functional groups or bonded phases. These modifications can tailor the column's selectivity, making it suitable for a wide range of analytes and applications. Common modifications include C18 (octadecyl), C8 (octyl), C4 (butyl), and others, each providing different selectivity.

Reproducibility: Silica-based HPLC columns offer good batch-to-batch reproducibility, which is crucial for obtaining consistent results in analytical and quality control laboratories.

Wide Applicability: Silica columns are versatile and can be used for the separation of a broad spectrum of compounds, including hydrophobic, polar, acidic, and basic analytes.

Scalability: Silica columns come in various particle sizes, allowing for scalability from analytical to preparative chromatography. Smaller particles provide higher resolution but higher backpressure.

Commercial Availability: Silica-based HPLC columns are readily available from numerous manufacturers in various dimensions and bonded phases, making it easy to find a column suitable for a specific application.

While silica is widely used and has many advantages, it's important to note that it may have limitations in specific applications. For example, silica can be susceptible to silanol interactions, which can lead to peak tailing and reduce resolution. To mitigate this issue, end-capping or alternative stationary phases (such as C8 or C18) are often used. Additionally, some biomolecules, like proteins, may adsorb to silica surfaces, necessitating the use of specialized columns or alternative stationary phases for their separation.

In summary, silica-based HPLC columns are an essential tool in analytical chemistry and are used in a wide range of applications due to their purity, inertness, and versatility. Researchers and analysts often select silica-based columns and modify them to suit their specific separation needs.

Normal Phase (NP): In normal phase chromatography, the stationary phase is polar and the mobile phase is non-polar. This type of column chemistry is used for the separation of non-polar and moderately polar analytes, such as lipids, steroids, and some natural products. Common stationary phases used in normal phase columns include silica particles coated with polar groups, such as diol (OH), cyano (CN), or amino (NH2) groups.

Ion Exchange (IE): In ion exchange chromatography, the stationary phase contains charged groups that interact with analytes based on their ionic properties. This type of column chemistry is commonly used for the separation of charged analytes, such as ions, amino acids, and proteins. Ion exchange columns can be either anion exchange (stationary phase with positively charged groups) or cation exchange (stationary phase with negatively charged groups).

Size Exclusion (SEC) or Gel Permeation (GPC): Size exclusion chromatography, also known as gel permeation chromatography, is used for the separation of analytes based on their size or molecular weight. The stationary phase in SEC columns typically consists of porous beads with defined pore sizes, which allow smaller analytes to enter the pores and take longer to elute, while larger analytes pass around the beads and elute faster.

Chiral Chromatography: Chiral chromatography is used for the separation of enantiomers, which are mirror-image isomers of a molecule that have different physiological activities. Chiral stationary phases can be either reverse phase or normal phase, and they contain chiral selectors that interact selectively with one enantiomer over the other, resulting in enantioseparation.

Affinity Chromatography: Affinity chromatography is used for the separation and purification of analytes based on their specific interactions with a stationary phase that contains ligands with high affinity for the analyte of interest. This type of column chemistry is commonly used in bioanalysis, such as protein purification or drug-protein binding studies.

These are some of the common types of column chemistries used in HPLC. The choice of column chemistry depends on the specific analytes to be separated, the sample matrix, and the desired separation characteristics, such as selectivity, efficiency, and resolution. Proper selection of column chemistry is crucial for achieving accurate and reliable results in HPLC analysis.

C18 Columns: 

C18 HPLC columns are a type of reverse phase chromatography column commonly used in high-performance liquid chromatography (HPLC). These columns are packed with a stationary phase that has octadecyl (C18) functional groups bonded to the silica surface. C18 columns are versatile and widely used for the separation of a wide range of analytes, particularly hydrophobic and organic compounds. There are several variations of C18 columns designed to meet specific separation requirements. Here are some types of C18 HPLC columns:

Conventional C18 Columns: These are the standard C18 columns with octadecyl functional groups bonded to the silica surface. They are suitable for a wide range of applications, including pharmaceuticals, environmental analysis, and more.

End-Capped C18 Columns: To improve peak shape and reduce secondary interactions with residual silanol groups on the silica surface, C18 columns are often "end-capped." End-capping involves treating the stationary phase to block these silanol groups, resulting in better peak symmetry and less tailing of peaks.

Wide-Pore C18 Columns: These columns have larger pore sizes in the silica matrix, which are designed for the separation of larger molecules such as proteins and peptides. Wide-pore C18 columns are commonly used in the analysis of biomolecules.

Ultra-High-Performance C18 Columns (UHPLC C18): UHPLC C18 columns are designed for use with ultra-high-performance liquid chromatography (UHPLC) systems, which operate at higher pressures and flow rates. These columns offer improved efficiency and faster separations compared to standard HPLC C18 columns.

Monolithic C18 Columns: Instead of the traditional packed bed of particles, monolithic C18 columns have a single, continuous piece of stationary phase. These columns offer high speed and efficient separations due to their unique structure.

Shielded C18 Columns: Shielded C18 columns are designed to minimize secondary interactions between the analytes and the silica surface. They have a shielding layer on the silica surface to reduce the impact of residual silanol groups.

Polar-Embedded C18 Columns: These columns have C18 functional groups modified with polar groups. They offer improved selectivity for polar compounds while retaining the advantages of C18 stationary phases for nonpolar compounds.

Mixed-Mode C18 Columns: Mixed-mode C18 columns incorporate additional interactions beyond hydrophobic interactions. They may have ion-exchange or other functional groups bonded to the C18 phase, allowing for unique selectivity for specific analytes.

Hydrophilic Interaction Liquid Chromatography (HILIC) C18 Columns: These columns combine the hydrophobic properties of C18 with the principles of HILIC chromatography, making them suitable for the separation of polar and hydrophilic compounds.

Chiral C18 Columns: Chiral C18 columns combine C18 functionality with chiral selectors for enantioselective separations, allowing the separation of enantiomers.

The choice of C18 column type depends on the specific analytes and separation requirements of your HPLC analysis. Each type of column offers unique advantages for different applications, and selecting the right one is essential for achieving the desired separation and chromatographic performance.

Types of C8 columns:

C8 HPLC columns are a type of reverse phase chromatography column that contain a stationary phase with octyl (C8) functional groups bonded to the silica surface. These columns are commonly used in high-performance liquid chromatography (HPLC) for the separation of a wide range of analytes, particularly hydrophobic and organic compounds. There are several variations of C8 columns designed to meet specific separation requirements. Here are some types of C8 HPLC columns:

Conventional C8 Columns: These are the standard C8 columns with octyl functional groups bonded to the silica surface. They are suitable for a wide range of applications, including pharmaceuticals, environmental analysis, and more.

End-Capped C8 Columns: To improve peak shape and reduce secondary interactions with residual silanol groups on the silica surface, C8 columns are often "end-capped." End-capping involves treating the stationary phase to block these silanol groups, resulting in better peak symmetry and less tailing of peaks.

Wide-Pore C8 Columns: These columns have larger pore sizes in the silica matrix, which are designed for the separation of larger molecules such as proteins and peptides. Wide-pore C8 columns are commonly used in the analysis of biomolecules.

Ultra-High-Performance C8 Columns (UHPLC C8): UHPLC C8 columns are designed for use with ultra-high-performance liquid chromatography (UHPLC) systems, which operate at higher pressures and flow rates. These columns offer improved efficiency and faster separations compared to standard HPLC C8 columns.

Shielded C8 Columns: Shielded C8 columns are designed to minimize secondary interactions between the analytes and the silica surface. They have a shielding layer on the silica surface to reduce the impact of residual silanol groups.

Mixed-Mode C8 Columns: Mixed-mode C8 columns incorporate additional interactions beyond hydrophobic interactions. They may have ion-exchange or other functional groups bonded to the C8 phase, allowing for unique selectivity for specific analytes.

Hydrophilic Interaction Liquid Chromatography (HILIC) C8 Columns: These columns combine the hydrophobic properties of C8 with the principles of HILIC chromatography, making them suitable for the separation of polar and hydrophilic compounds.

Chiral C8 Columns: Chiral C8 columns combine C8 functionality with chiral selectors for enantioselective separations, allowing the separation of enantiomers.

The choice of C8 column type depends on the specific analytes and separation requirements of your HPLC analysis. Each type of column offers unique advantages for different applications, and selecting the right one is essential for achieving the desired separation and chromatographic performance.